Antibiotic A16886II

ABSTRACT

Antibiotic A16886I and antibiotic A16886II and their salts, having antibacterial and anthelmintic activity, prepared by fermentation of Streptomyces clavuligerus NRRL 3585.

CROSS-REFERENCE TO RELATED APPLICATION

This is a division of application Ser. No. 463,233, filed Apr. 23, 1974,which was in turn a division of pending application Ser. No. 62,390,filed Aug. 10, 1970. Application Ser. No. 62,390 was acontinuation-in-part of then pending application Ser. No. 849,395, filedAug. 12, 1969, and abandoned after the filing of application Ser. No.62,390.

SUMMARY OF THE INVENTION

Fermentation of Streptomyces clavuligerus produces new antibioticsubstances designated herein as antibiotic A16886. The salts ofantibiotic A16886 are readily obtained by reaction of antibiotic A16886with a suitable acid or base. Antibiotic A16886 and its salts exhibitantibacterial and anthelmintic activity. The antibacterial activity isexhibited primarily against gram-negative organisms, but some activityis also exhibited against gram-positive organisms. Antibiotic A16886 isalso effective in controlling disease caused by plant-pathogenicbacteria.

DESCRIPTION OF PREFERRED EMBODIMENTS

Antibiotic A16886, a sulfur-containing peptide antibiotic, is anamphoteric molecule produced by cultivating under controlled conditionsStreptomyces clavuligerus NRRL 3585.

As in the case with many antibiotic-producing cultures, fermentation ofStreptomyces clavuligerus NRRL 3585 results in the production of anumber of antibiotic substances called "factors." Antibiotic A16886, aspresently obtained from the fermentor, comprises two major antibioticsubstances referred to herein as A16886I and A16886II or as factor I andfactor II. Other antibiotic substances have been observed in somefermentations, but these have been present in only small amounts. Hence,the unmodified term "antibiotic A16886" is employed herein to refer tothe antibiotic as obtained from the fermentor and ordinarily comprisingfrom 1 to 99 percent of factor I and from 99 to 1 percent of factor IIin addition to other minor factors, the total percentage being 100.Accordingly, in the discussions herein of utility and modes of usage,the term "antibiotic A16886" applies equally to individual factors aswell as to mixtures inasmuch as the properties of the individual factorsand mixtures thereof are similar.

Antibiotic A16886 can be utilized as such or as a salt, for example, anacid addition salt or a salt with a cation. In the instance of a saltwith a cation, the salt can be either a mono or di salt. It is oftenpreferred to prepare salts directly in the purification process so thatthe antibiotic as separated is in salt form. Antibiotic A16886 has beenseparated in this manner, as the monoammonium salt, and for that reason,is hereinbelow characterized as the monoammonium salt.

Certain characteristics have been made on the mixture of factors I andII. For example, a number of qualitative chemical tests have beencarried out with a mixture of the monoammonium salt of factor I and themonoammonium salt of factor II: positive tests were exhibited withninhydrin, Pan-Dutscher, Benedict, Molisch, Fehling, dansyl chloride,iodine, and ferric chloride reagents, but negative tests were observedwith biuret and Sakaguchi reagents. A mixture of the monoammonium saltsof factors I and II dried at room temperature in vacuo over anhydrouscalcium chloride for about 15 hours, exhibited an optical rotation[α]_(D) ²⁵ + 110.1° (C = 1 percent w./v. in water). A mixture of themonoammonium salts of factors I and II of antibiotic A16886 is stable atpH 3-8 at 6° C. for 8 days and relatively stable at pH 3-8 at 25° C. for2 days. Biological activity is slowly lost at pH 3-8 at a temperature of25° C., half being lost at 4 days.

In addition, the monoammonium salts of factors I and II have beenseparately characterized. The monoammonium salt of antibiotic A16886I isan off-white, fluffy, amorphous solid, softening between 190° and 300°C., with a color change to dark brown; it is very soluble in water,slightly soluble in lower alkanols, and essentially insoluble inacetonitrile and other organic solvents. The specific optical rotation[α]_(D) ²⁵ of the monoammonium salt of antibiotic A16886I, dried at roomtemperature in vacuo over anhydrous calcium chloride for about 15 hours,was found to be +153.6° (C = 1 percent, w./v. in water). Electrometrictitration of the monoammonium salt of antibiotic A16886I in a 66 percentdimethylformamide-water solution at an initial pH of 6.50 revealed thepresence of four titratable groups: pK'a₁ = 4.1; pK'a₂ = 5.2; pK'a₃ =9.3; and pK'a₄ = 10.5. The apparent molecular weight, calculated fromthe titration data, is about 530.

Elemental analysis of the monoammonium salt of A16886I, dried in vacuoat about 80° C. over phosphorus pentoxide, gave the following values:

    ______________________________________                                               Element       Percent                                                  ______________________________________                                               Carbon        40.26                                                           Hydrogen       5.91                                                           Nitrogen      13.98                                                           Oxygen        33.37                                                           Sulfur         7.21                                                    ______________________________________                                    

Analysis of the monoammonium salt of antibiotic A16886I shows a methoxylcontent of 5.02 percent; the presence of the methoxyl group wasconfirmed by a singlet at 3.53 ppm. in the NMR spectrum. A Van Slyketest for amino nitrogen of the monoammonium salt of antibiotic A16886Ishowed 5.3 percent.

The NMR spectrum of A16886I in D₂ O showed the followingcharacteristics: 5.19 ppm. (1H, singlet); 4.94, 4.74 ppm. (2H, ABquartet, J = 13 Hz); 4.0-4.2 ppm. (1H, multiplet); 3.68, 3.32 ppm. (2H,AB quartet, J = 18 Hz); 3.53 ppm. (3H, singlet); 2.6-2.4 ppm. (2H,multiplet); 2.1-1.6 ppm. (4H, multiplet).

The infrared absorption spectrum of the monoammonium salt of antibioticA16886I in a mineral oil mull is shown in FIG. 1 of the accompanyingdrawings. The distinguishable bands in the infrared spectrum over therange of 2.0 to 15.0 microns are as follows: 3.10, 5.68, 6.30, 6.60,7.20, 7.60, 8.83, 9.35, 9.60, 9.85, 11.60, and 12.90 microns.

The ultraviolet absorption spectrum of the monoammonium salt ofantibiotic A16886I in aqueous solution shows absorption maxima at 242 mμ(E₁ cm^(1%) = 132) and at 264 mμ (E₁ cm^(1%) = 165); circular dichroismwas also measured in aqueous solution and showed a positive Cottoneffect at 263 mμ and a negative Cotton effect at 236 mμ.

Paper chromatography of the ammonium salt of antibiotic A16886I ofWhatman No. 1 paper gave an R_(f) value of 0.41 in a solvent system ofpropanol, acetonitrile, and water in a volume ratio of 1:1:1.Bioautographs were obtained by placing the paper chromatograph on agarplates seeded with Salmonella gallinarum as the test organism.

When the monoammonium salt of A16886I is subjected to thin-layerchromatography on silica gel plates in 70 percent aqueous acetonitrile,utilizing a ninhydrin spray as a dector, it has an R_(f) value of about0.51; on cellulose plates in acetonitrile:isopropanol:water (1:1:1),utilizing the same procedure for detection, it has an R_(f) value of0.36.

Amino acid analysis of an acid hydrolysate of antibiotic A16886I, run bythe Spackman-Moore-Stein technique, showed two ninhydrin reacting peaks,one of which was eluted identically with glycine (0.61 μmoles/mg.), theother of which was eluted just prior to glycine and was identified asα-aminoadipic acid (1.97 μmoles/mg.).

Based on the various foregoing physical characteristics the structure ofantibiotic A16886I has been determined to be as follows: ##STR1## whichstructure is designated7-(5-amino-5-carboxyvaleramido)-3-(carbamoyloxymethyl)-7-methoxy-3-cephem-4-carboxylicacid.

The monoammonium salt of antibiotic A16886II has also been separatelycharacterized. It is an off-white, fluffy, amorphous solid, softeningbetween 190° and 300° C., with a color change to dark brown; it is verysoluble in water, slightly soluble in lower alkanols, and essentiallyinsoluble in acetonitrile and other organic solvents. The specificoptical rotation [α]_(D) ²⁵ of the monoammonium salt of antibioticA16886II, dried at room temperature in vacuo over anhydrous calciumchloride for about 15 hours, was found to be +86.2° (C = 1 percent,w./v. in water).

Electrometric titration of the monoammonium salt of antibiotic A16886IIin a 66 percent dimethyl formamide-water solution at an initial pH of7.7, revealed the presence of four titratable groups: pK'a₁ = 4.4; pK'a₂= 5.7; pk'a₃ = 9.6; pK'a₄ = 10.4. In like titration of a later sample,except that it showed an initial pH of 6.8, the respective values werepK'a₁ = 4.0; pk'a₂ = 5.3; pK'a₃ = 9.2; and pK'a₄ = 10.5. The apparentmolecular weight of the monoammonium salt of antibiotic A16886II,calculated from the titration data, is about 528.

Elemental analysis of the monoammonium salt of antibiotic A16886II,dried in vacuo at about 80° C. over phosphorus pentoxide, gave thefollowing values:

    ______________________________________                                               Element       Percent                                                  ______________________________________                                               Carbon        41.01                                                           Hydrogen       5.64                                                           Nitrogen      15.75                                                           Oxygen        29.28                                                           Sulfur         7.04                                                    ______________________________________                                    

Analysis of the monoammonium salt of A16886II showed no methoxylcontent, and, unlike factor I, no signal attributable to the methoxylgroup was seen in the NMR spectrum on the monoammonium salt of A16886II.A Van Slyke test for amino nitrogen on the monoammonium salt ofantibiotic A16886II showed 5.1 percent.

The NMR spectrum of A16886II in D₂ O showed the followingcharacteristics: 5.67 ppm. (1H, doublet, J = 5 Hz); 5.15 ppm (1H,doublet, J = 5 Hz); 4.90, 4.68 ppm. (2H, AB quartet, J = 13 Hz); 3.9-3.7(1H, multiplet); 3.69, 3.39 (2H, AB quartet, J = 18 Hz); 2.6-2.3 (2H,multiplet); 2.1-1.5 (4H, multiplet).

The infrared absorption of the monoammonium salt of antibiotic A16886IIin a mineral oil mull is shown in FIG. 2 of the accompanying drawings.The distinguishable bands in the infrared spectrum over the range of 2.0to 15.0 microns are as follows: 3.15 5.70, 6.30, 7.22, 7.64, 9.05, 9.40,9.65, and 11.60 microns.

The ultraviolet absorption spectrum of the monoammonium salt ofantibiotic A16886II in aqueous solution shows an absorption maximum of260 mμ (E₁ cm^(1%) = 148); circular dichroism was also measured inaqueous solution and showed a positive Cotton effect at 258 mμ and anegative Cotton effect at 228 mμ.

Paper chromatography of the monoammonium salt of antibiotic A16886II onWhatman No. 1 paper gave an R_(f) value of 0.33 in a solvent system ofpropanol, acetonitrile, and water in a volume ratio of 1:1:1.Bioautographs were obtained by placing the paper chromatograph on agarplates seeded with Salmonella gallinarum as the test organism.

When the monoammonium salt of A16886II is subjected to thin-layerchromatography on silica gel plates in 70 percent aqueous acetonitrile,utilizing a ninhydrin spray or bioautographic method as a detector, ithas an R_(f) value of about 0.42; on cellulose plates inacetonitrile:isopropanol:water (1:1:1), utilizing the same procedure fordectection, it has an R_(f) value of 0.29.

Amino acid analysis of an acid hydrolysate of antibiotic A16886II, runby the Spackman-Moore-Stein technique, showed primarily only oneninhydrin reacting peak; it was eluted just prior to glycine and as inthe instance of antibiotic A16886I, was identified as α-aminoadipic acid(2.1 μmoles/mg.). However, there was also a very minor peak which waseluted identically with glycine. A later sample was analyzed in likemanner and showed values of 2.1 μmoles/mg. and 0.13 μmoles/mg.,respectively.

Based on the various foregoing physical characteristics, the structureof antibiotic A16886II has been determined to be as follows: ##STR2##which structure is designated7-(5-amino-5-carboxyvaleramido)-3-(carbamoyloxymethyl)-3-cephem-4-carboxylicacid.

In addition to the foregoing, each of factors I and II of themonoammonium salt of antibiotic A16886 was subjected to paperchromatography and thin-layer chromatography in a number of othersolvent systems with the following results:

    ______________________________________                                         Solvent System     R.sub.f value                                             ______________________________________                                        Paper Chromatography                                                                             Factor 1   Factor II                                       ______________________________________                                        Ethanol:water (80:20) with 1.5%                                               sodium chloride, paper impregnated                                            with 1N sodium sulfate                                                                           .38        .33                                             Butanol saturated with water                                                                     immobile   immobile                                        Butanol saturated with water                                                  plus 2% p-toluenesulfonic acid                                                                   .39        .32                                             Methyl isobutyl ketone saturated                                              with water         immobile   immobile                                        Methyl isobutyl ketone saturated                                              with water plus 2% p-toluene-                                                 sulfonic acid      immobile   immobile                                        Methyl isobutyl ketone saturated                                              with water plus 2% piperidine                                                                    immobile   immobile                                        Acetonitrile       immobile   immobile                                        Propanol:acetonitrile:methanol:                                               water (4:3:2:1)    immobile   immobile                                        Propanol:pyridine:acetic acid:                                                water (15:10:3:12) .32        .27                                             Propanol:pyridine:acetic acid:                                                acetonitrile:water (45:30:9:40:36)                                                               .21        .15                                             Butanol:acetic acid:water (3:1:1)                                                                .20        .17                                             Ethyl acetate:acetic acid:water                                               (3:1:1)            .29        .22                                             Propanol:water (70:30)                                                                           .17        .17                                             Acetonitrile:water (70:30)                                                                       .72        .65                                             Water:ethanol:acetic acid (70:42:6)                                                              .82        .82                                             Thin-Layer Chromatography                                                     Acetonitrile:water (7:3) on                                                   cellulose plates   .35        .29                                             ______________________________________                                    

Antibiotic A16886 and its salts have an inhibitory action against thegrowth of both gram-positive and gram-negative bacteria. The levels atwhich antibiotic A16886 as a partially purified mixture of themonoammonium salts of factors I and II shows inhibition against thegrowth of illustrative organisms are set forth in Table I. Theinhibitory levels were determined by the agardilution test or by thebroth-dilution test (identified in the table by the letters "a.d." and"b.d.", respectively).

In the agar dilution test, the test organism was streaked on a series ofagar plates containing various concentrations of the monoammonium saltsof factors I and II of antibiotic A16886 to determine the minimumconcentrations in meg./ml. (micrograms per milliliter) in the agarsubstrate which inhibited the growth of the organism over a period offorty-eight hours (seventy-two hours in the case of the plant-pathogenorganisms).

In the broth dilution test, a series of tubes containing variedconcentrations of the monoammonium salts of factors I and II ofantibiotic A16886 was inoculated with the test organism to determine theminimum concentration in mcg./ml. in the broth substrate which inhibitedorganism growth for a period of about twenty hours.

                  TABLE I                                                         ______________________________________                                                           Inhibitory Concentration                                   Test Organism      mcg./ml.                                                   ______________________________________                                        Escherichia coli 0127                                                                            0.39 b.d.                                                  Proteus PR-6       0.78 a.d.                                                  Salmonella typhimurium S-4                                                                       0.78 a.d.                                                  Klebsiella sp. K-1 1.56 a.d.                                                  Pseudomonas sp. X239                                                                             >100 a.d.                                                  Salmonella typhosa T-63                                                                          0.78 b.d.                                                  Staphylococcus aureus 3055                                                                       25.00 a.d.                                                 Streptococcus pyogenes C203                                                                      6.25 a.d.                                                  Bacillus subtilis X12.1                                                                          1.56 a.d.                                                  Staphylococcus aureus 3150                                                                       50.00 a.d.                                                 ______________________________________                                    

No binding by horse serum was noted in any of the above tests. As can beseen from the above table, antibiotic A16886 as a mixture of themonoammonium salts of factors I and II exhibits activity againstgram-positive and gram-negative bacterial organisms.

Antibiotic A16886 was further evaluated for antibacterial activity in atest employing the Bauer-Kirby disc diffusion method. The evaluation wasmade on the monoammonium salts of separated factors I and II. Theresults, expressed in terms of millimeters of the zone of inhibition ata specified antibiotic concentration, were as set forth below in TableII.

                                      TABLE II                                    __________________________________________________________________________                  Factor I           Factor II                                                  μg./disc       μ g./disc                                  Organism      30 10 5  2.5                                                                              1.0                                                                              0.5                                                                              30 10 5  2.5                                                                              1.0                                                                              0.5                            __________________________________________________________________________    Escherichia coli 0127                                                                       25.6                                                                             20.7                                                                             18.6                                                                             15.9                                                                             12.7                                                                             8.4                                                                              19.7                                                                             15.9                                                                             12.9                                                                             9.4                                                                              0  0                              Escherichia coli EC 25                                                                      19.6                                                                             15.1                                                                             12.5                                                                             9.1                                                                              0  0  11.9                                                                             7.7                                                                              0  0  0  0                              Proteus sp. PR6                                                                             23.2                                                                             19.0                                                                             16.7                                                                             13.6                                                                             9.9                                                                              0  22.2                                                                             16.7                                                                             13.6                                                                             10.4                                                                             0  0                              Proteus sp. PR7                                                                             14.0                                                                             10.4                                                                             0  0  0  0  7.1                                                                              0  0  0  0  0                              Salmonella typhosa SA 12                                                                    27.2                                                                             22.8                                                                             19.9                                                                             16.9                                                                             11.3                                                                             8.0                                                                              23.8                                                                             18.7                                                                             15.6                                                                             12.3                                                                             7.5                                                                              0                              Salmonella typhosa SA 16                                                                    24.2                                                                             19.6                                                                             16.2                                                                             13.1                                                                             9.3                                                                              0  19.3                                                                             13.4                                                                             11.0                                                                             7.5                                                                              0  0                              Klebsiella-Aerobacter KA 14                                                                 23.0                                                                             18.8                                                                             15.4                                                                             11.4                                                                             7.6                                                                              0  20.1                                                                             13.7                                                                             10.8                                                                             0  0  0                              Klebsiella-Aerobacter KA 25                                                                 22.1                                                                             15.8                                                                             12.3                                                                             9.0                                                                              0  0  15.6                                                                             10.1                                                                             7.4                                                                              0  0  0                               Pseudomonas sp. Ps 24                                                                      23.0                                                                             18.2                                                                             15.7                                                                             13.2                                                                             10.1                                                                             0  20.2                                                                             15.4                                                                             12.3*                                                                            9.3*                                                                             0  0                              Pseudomonas sp. Ps 30                                                                       0  0  0  0  0  0  0  0  0  0  0  0                              Staphylococcus aureus 3055                                                                  0  0  0  0  0  0  12.4                                                                             0  0  0  0  0                              Staphylococcus aureus 3074                                                                  0  0  0  0  0  0  10.1                                                                             0  0  0  0  0                              __________________________________________________________________________     *Satellite colonies within zone of inhibition                            

Antibiotic A16886 and its salts also exhibit in vivo activity against anumber of the above organisms and hence are useful in controllinginfections caused by such organisms in host animals. Mammalian toxicityis low; the LD₅₀ is > 5 grams/kilogram, and daily subcutaneousadministration of 350 milligrams/kilogram to a group of 10 rats for aperiod of fourteen days resulted in no deaths and negligible signs oftoxicity. Antibiotic A16886 as a mixture of the monoammonium salts offactors I and II exhibited the following ED₅₀ values in mice infectedwith the respective organism:

                  TABLE III                                                       ______________________________________                                        Organism       ED.sub.50                                                      ______________________________________                                        Escherichia coli 0127                                                                        13.8     (Administration was by                                                        the sub-cutaneous                                     Salmonella typhosa T-63                                                                      15.6     route)                                                Klebsiella K-1 10.2                                                           Proteus PR-6    7.3                                                           ______________________________________                                    

In addition, antibiotic A16886 and its salts exhibit activity againstplant-pathogenic bacteria. Thus, for example, antibiotic A16886 and itssalts control such plant-pathogenic bacterial infections as bacterialwilt, bacterial spot, and bacterial blight.

The precise manner of application of antibiotic A16886 or a salt thereofto plants is not critical. Generally, initial contact of the causativeorganism is with plant foliage; for this reason, foliar application ofantibiotic A16886 or a salt thereof is often preferred. However,antibiotic A16886 and salts thereof are translocated; consequently,application can also be made to stems, flowers, seeds, roots, or otherplant parts to obtain bactericidal effect throughout plants.

The present method for controlling plant-pathogenic bacteria comprisesapplying to a plant exposed to the bacteria an effective amount ofantibiotic A16886 or a salt thereof. It is not critical to this methodthat the antibiotic be employed as a single factor; each of factor I andfactor II can be used alone and gives good results. Alternatively, amixture of factors can be employed. Antibiotic A16886 or a salt thereofcan be employed without modification, but generally it is preferred forplant-treating purposes to employ a formulation comprising theantibiotic A16886 or a salt thereof and one or more adjuvants. Inpreparing such formulations, antibiotic A16886 or its salt can bemodified with water or other liquid carriers, organic solvents,surface-active agents including solid surface-active agents, or inert,finely divided solids or inert, granular solids. The exact concentrationof antibiotic A16886 or a salt thereof in such a formulation is notcritical and will vary with the particular purpose for which theformulation is designed. Generally, for foliar application for controlof typical plant-pathogenic bacterial infections, concentrations ofantibiotic A16886 or a salt thereof from 10 ppm. to 1000 or more ppm.give good results. In the case of dusts for foliar application,preferred concentrations of antibiotic are 0.1-10.0 percent.

In preparing liquid formulations, antibiotic A16886 or a salt thereofcan be compounded with a suitable liquid and a surface-active dispersingagent to produce emulsifiable concentrates which can be further dilutedwith water or other liquid to form spray mixtures. Preferred dispersingagents which can be employed in these compositions include the non-ionicemulsifiers such as the condensation products of alkylene oxides withthe inorganic acids, polyoxyethylene derivatives of sorbitan esters,complex ether alcohols, and the like. Suitable organic liquids which canbe employed in the composition include petroleum oils and distillates,toluene, and synthetic organic oils. The surface-active dispersingagents are usually employed in liquid compositions in the amount of from0.1 to 20 percent by weight of the combined weight of the dispersingagent and active compound. In the preparation of dust compositions,antibiotic A16886 or a salt thereof can be compounded with any of thefinely divided solids or granules typically used in agriculturalchemical formulations.

When operating in accordance with the present invention, antibioticA16886 or a salt thereof or a composition containing antibiotic A16886or a salt thereof can be applied to the organisms to be controlled, orto their habitats in any convenient fashion, e.g., by means of handdusters or sprayers. Applications to the above-ground portions of plantsconveniently can be carried out with powder dusters, boom sprayers,high-pressure sprayers, and spray dusters. In foliar applications, theemployed compositions should not contain any appreciable amount ofphytotoxic diluents. In large-scale operations, dusts or low-volumesprays can be applied from airplanes.

The following examples illustrate this embodiment of the presentinvention and will enable those skilled in the art to practice the same.

EXAMPLE 1 ERWINIA AMYLOVORA AND PSEUDOMONAS SOLANACEARUM, IN VITRO

Antibiotic A16886 (as a mixture of the monoammonium salt of factor I andthe monoammonium salt of factor II) was evaluated in vitro forinhibition of Erwinia amylovora and Pseudomonas solanacearum. Each ofthe organisms was separately incorporated in standard nutrient agar, andthe resulting agars poured into a plurality of plates and permitted tosolidify. Treatment was then made, different plates being treated bydifferent methods. In one method of treatment, 0.1 milliliter of testsolution was pipetted into each of two small steel cylinders settingupright on a given agar surface. In the other method, 0.1 milliliter oftest solution was impregnated onto each of two filter assay discs whichwere placed onto a given agar surface. The test solution for bothmethods of treatment was prepared by dissolving the appropriate quantityof antibiotic A16886 in glass-distilled water. The results, expressed asthe diameter of the zone of inhibition, were as set forth in thefollowing table:

                                      TABLE IV                                    __________________________________________________________________________    DIAMETER - ZONE INHIBITION MEASUREMENT IN MILLILITERS                                     Steel Cylinders.sup.A                                                                         Filter Discs.sup.B                                Treatment   E. amylovora                                                                         P. solanacearum                                                                        E. amylovora                                                                         P. solanacearum                            __________________________________________________________________________    A16886 - 10 ppm.                                                                           6-6   16-17    13-13  20-21                                      A16886 - 50 ppm.                                                                          12-14  25-25    16-17  30-30                                      A16886 - 100 ppm.                                                                         15-16  30-30    20-20  34-35                                      Control (distilled water)                                                                 6-6    6-6      13-13  13-13                                      __________________________________________________________________________     .sup.A includes the size of cylinder - 6mm.; therefore, a zone size of 6      mm. indicates no growth inhibition.                                           .sup.B includes the size of filter disc - 13 mm.; therefore, a zone size      of 13 mm. indicates no growth inhibition.                                

EXAMPLE 2 PSEUDOMONAS SOLANACEARUM, FOLIAR SPRAY

Antibiotic A16886 (as a mixture of the monoammonium salt of factor I andthe monoammonium salt of factor II) was also evaluated in vivo for thecontrol of Pseudomonas solanacearum on tomato plants. In thisevaluation, antibiotic A16886 monoammonium salt was formulated in aplurality of aqueous formulations uniformly containing 1 percent ofethanol and 0.1 percent of a surface-active agent (a polyoxyethylenederivative of fatty acid partial ester of sorbitol anhydride) butcontaining various concentrations of antibiotic A16886. Thirty-day-oldtomato plants were used in the evaluation, 2 plants/pot/treatment.Plants in each pot were treated with one of the treating solutions, wereallowed to air dry, and were then inoculated with a medium sustaining anactive growth of Pseudomonas solanacearum. All plants were held for 24hours in a moist chamber, then removed and held for 7 days under goodagricultural conditions. At the end of this seven-day period, all of theplants were observed to determine presence, and if present, degree, ofinfection. The results were as reported in the following table; diseasewas rated on a scale of 0 to 4, 0 indicating no control, 4 representingcomplete control, with each plant rated separately.

                  TABLE V                                                         ______________________________________                                                          Pseudomonas solanacearum                                                     Disease Rating                                               ______________________________________                                        Control (1% EtOH + 0.1%                                                       surface-active agent)                                                                            0          0                                               A16886 - 400 ppm.   2+         3+                                             A16886 - 200 ppm.   2+         2+                                             ______________________________________                                    

The control plants exhibited an extensive Pseudomonas solanacearuminfection.

EXAMPLE 3 PSEUDOMONAS GLYCINIA, BARE-ROOT SOAK

Antibiotic A16886 (as a mixture of the monoammonium salt of factor I andthe monoammonium salt of factor II) was evaluated for the control ofPseudomonas glycinia by means of bare-root soaking solutions containingthe A16886. There were two soaking solutions, one containing 200 partsper million of antibiotic A16886, the other containing 400 parts permillion of antibiotic A16886. Ten-day-old soybean plants were inoculatedwith Pseudomonas glycinia, and the roots of all plants maintainedthroughout the evaluation in aqueous solutions. Initially, theinoculated plants were placed in 50 milliliters of solution, one groupof plants in the 200 ppm. solution, a second group of plants in the 400ppm. solution, a third group of plants in water to serve as a control.The roots were soaked for 24 hours, with aeration during this period.The plants, with roots still in the several solutions, were thenincubated for twenty-four hours. Thereafter, the plants were removedfrom the incubation chamber and held for a period of a week. Beginningapproximately with the incubation period and continuing throughout theweek, a solution containing nutrients was added to the roots as neededto maintain the plants in a viable condition. At the end of the weekperiod, the plants were observed to determine the extent of developmentof Pseudomonas glycinia; disease was rated on a scale of 0 to 4, 0indicating no control, 4 representing complete control. The results wereas set forth in the following table:

                  TABLE VI                                                        ______________________________________                                                         Disease Rating of                                                             Pseudomonas glycinia                                         ______________________________________                                        Control            0                                                          A16886 - 200 ppm.   3+                                                        A16886 - 400 ppm.   2+                                                        ______________________________________                                    

EXAMPLE 4 XANTHOMONAS PHASEOLI VAR. SOJENSIS, FOLIAR SPRAY

In a first evaluation, ten-day-old soybean plants were inoculated bylightly water-soaking the lower leaf surfaces of the primary leaves witha suspension of Xanthomonas phaseoli var. sojensis. Two hours later, thefoliage was sprayed with a spray solution, one group of inoculatedplants being treated with a solution containing 100 parts per million ofantibiotic A16886, a second group, with a solution containing 500 partsper million of antibiotic A16886. Antibiotic A16886 was formulated inwater with 353 parts per million of a blend of sulfonate-nonionic liquidemulsifiers.

In a second evaluation, the foliage of ten-day-old soybean plants wassprayed with a spray solution containing either 100 or 500 parts ofantibiotic A16886, and, two hours later, inoculated by the sameprocedures used in the first evaluation. Formulation of antibioticA16886 was the same as in the first evaluation. All samples ofantibiotic A16886 were a mixture of the monoammonium salt of factor Iand the monoammonium salt of factor II.

The plants were maintained under good agricultural conditions for aperiod of about 11 days and then observed for the presence or absence ofXanthomonas phaseoli var. sojensis. The results were as set forth in thefollowing table, with rating being on a 0 to 4 scale as in the precedingexamples.

                  TABLE VII                                                       ______________________________________                                                      Disease Rating Average Per                                                    Treated Group of Plants                                                       Xanthamonas phaseoli var. sojensis                                            Inoculate spray                                                               then spray                                                                              then inoculate                                        ______________________________________                                        Control (water alone)                                                                         0           0                                                 Control (water plus blend of                                                  emulsifier)     0           0                                                 A16886 - 100 ppm.                                                                             2           4                                                 A16886 - 500 ppm.                                                                              3+         4                                                 ______________________________________                                    

EXAMPLE 5 PSEUDOMONAS PHASEOLICOLA, FOLIAR SPRAY

All surfaces of twenty-day-old red kidney bean plants were sprayed withan aqueous formulation containing 400 parts per million of antibioticA16886 (as a mixture of the monoammonium salt of factor I and themonoammonium salt of factor II), and 0.1 percent of a surface-activeagent (a polyoxyethylene derivative of a fatty acid partial ester ofsorbitol anhydride). The sprayed plants were permitted to dry and theninoculated with Pseudomonas phaseolicola inoculation was achieved bywater-soaking of the lower leaf surface of one leaflet of each of thefirst and second trifoliate leaves with an aqueous bacterial suspensionat 30 percent light transmission on a Bausch and Lomb Spectronic 20. Theplants were subsequently held in a moist chamber for twenty-four hours,then removed and held under good agricultural conditions for 14 days. Atthe end of this period, all plants were observed; in the treated groupof plants, there was a moderate control of Pseudomonas phaseolicola,whereas in the control group, there was an extensive development of thedisease.

EXAMPLE 6 PSEUDOMONAS SOLANACEARUM, SEPARATE FOLIAR OR STEM SPRAY

Antibiotic A16886 (as a mixture of the monoammonium salt of factor I andthe monoammonium salt of factor II) was formulated in water with a blendof two sulfonate-nonionic liquid emulsifiers to obtain two treatingsolutions, one containing 100 parts per million of A16886, the othercontaining 400 parts per million of antibiotic A16886, and eachcontaining 353 parts per million of the emulsifier blend. Groups oftwenty-eight-day-old tomato plants were employed; in one set ofevaluations, the solution was applied to the stem only, and in thesecond set of evaluations, the solution was applied to the leaves only.Two hours after treatment by either method, the plants were inoculatedby inserting a toothpick soaked in a broth culture of Pseudomonassolanacearum into the stem at the cotyledons. The plants were held undergood agricultural conditions for 10 days and then observed to determinecontrol of the disease. The results, on a scale of 0-4, with 0indicating no control and 4 indicating complete control, were asfollows:

                  TABLE VIII                                                      ______________________________________                                                      Pseudomonas                                                                   solanacearum Disease Rating                                                   Leaves,        Stems,                                                         only   treated only    treated                                  ______________________________________                                        Control (water plus 353                                                       ppm. of blend of emulsifiers)                                                                 0        1       0     1                                      A16886 - 100 ppm.                                                                             3        4       3     4                                      A16886 - 400 ppm.                                                                             4        4       4     4                                      ______________________________________                                    

EXAMPLE 7 PSEUDOMONAS SOLANACEARUM, FOLIAR SPRAY

Further evaluations were conducted in accordance with the procedurereported in Example 2, except that antibiotic A16886I and A16886II wereevaluated separately (but each as the monoammonium salt). The resultswere as set forth in the following table.

                  TABLE IX                                                        ______________________________________                                                      Pseudomonas solanacearum                                                      Disease Rating                                                  ______________________________________                                        Control         0                                                             A16886I - 100 ppm.                                                                            4                                                             A16886I - 10 ppm.                                                                             2                                                             A16886II - 100 ppm.                                                                           4                                                             A16886II - 10 ppm.                                                                            1                                                             ______________________________________                                    

The control plants exhibited a light infection of Pseudomonassolanacearum.

EXAMPLE 8 PSEUDOMONAS SOLANACEARUM, FOLIAR SPRAY

A16886I monoammonium salt was evaluated again in the procedure ofExample 2 at concentrations in the treating solution of 100, 200, and400 ppm. of A16886I. A rating of 4 was obtained at each of theconcentrations. There was a heavy infestation of Pseudomonassolanacearum, on the control plants.

EXAMPLE 9 XANTHOMONAS VESICATORIA, FIELD TRIAL

A mixture of the monoammonium salt of A16886I and the monoammonium saltof A16886II was tested under field conditions for the control ofXanthomonas vesicatoria (bacterial spot) on mature bearing tomato plant.The mixture of the two salts was dispersed in water at a concentrationof 100, 200, or 400 parts per million and applied to the plants at therate of 75 gallons per acre. Application was made with a CO₂ sprayer at50 psi. Plants were sprayed twice, on successive days; after the secondspray had dried, all plants were wounded by sand blasting and thenwithin one minute the plants were inoculated with a bacterial inoculumof Xanthomonas vesicatoria. Three days later, a third spray applicationof the aqueous formulation of A16886 was made. Readings were made oneweek and two weeks after the last spray treatment. Controls consisted ofgroups of plants (1) sand blasted and inoculated; (2) sand blasted only;(3) untreated; and (4) treated with streptomycin (on the same three-dayspraying schedule, at a concentration of 100, 200, or 400 parts permillion of aqueous solution, using a rate of 75 gallons per acre), sandblasted, and inoculated. The results were as set forth in the followingtable, in which crop injury is reported on a scale of 0-10, with 0 = noinjury and 10 = dead plants; and control of bacterial spot is reportedon a percentage basis.

                                      TABLE X                                     __________________________________________________________________________                  Concentrations in                                                                      Crop Injury Ratings                                                                     Percent Bacterial Spot Control                             Parts/Million of                                                                       First                                                                              Second                                                                             First  Second                                              Treating Solution                                                                      Reading                                                                            Reading                                                                            Reading                                                                              Reading                               __________________________________________________________________________    A16886        100      0    0    88     46                                    A16886        200      0    0    85     63                                    A16886        400      0    0    97     80                                    Streptomycin  100      0    0    83     57                                    Streptomycin  200      0    0    81     37                                    Streptomycin  400      0    0    83     59                                    Control (Sand blasted and                                                     inoculated)    0       0    0    0 (14)*                                                                              0 (47)*                               Control (Sand blasted only)                                                                  0       0    0    0 (0.3)*                                                                             0 (8)*                                Control (Untreated)                                                                          0       0    0    0 (0)* 0 (6)*                                __________________________________________________________________________     *Parenthesized figures indicate percent disease incidence in the various      control plots. Disease incidence in the sand blasted and inoculated           control was used as the basis for the calculation of percent disease          control in the plants treated with Streptomycin and with A16886.         

As noted hereinabove, antibiotic A16886 and its salts exhibitanthelmintic activity in addition to antibacterial activity. Hence,antibiotic A16886 or a salt thereof can be administered to warm-bloodedanimals to control various internal parasites, particularly stomach andintestinal worms such as Ascaris lumbricoides var. suum, Aspiculuristetraptera, Syphacia obvelata, and the like. As in the instance ofbactericidal activity, anthelmintic activity is exhibited by each majorfactor hence, anthelmintic activity is obtained when employing eitherfactor separately, or when employing a mixture of factors.

The administration of antibiotic A16886 or a salt thereof is preferablyby the oral route, for example, by inclusion of antibiotic A16886 or asalt thereof in animal feed, by administration of tablets, drenches,etc. In general, doses of from 1 to 500 milligrams per kilogram or moreof animal body weight are effective in single-dose administration. Whereantibiotic A16886 or a salt thereof is supplied as a constituent of aregular feed, concentrations of from 0.0001 to 0.05 percent or more givegood results. A preferred range of concentration of antibiotic A16886 ora salt thereof in feeds is from 0.01 to 0.05 percent.

The anthelmintic activity of antibiotic A16886 is illustrated by thefollowing examples.

EXAMPLE 10

In a first evaluation, antibiotic A16886 (as a mixture of themonoammonium salt of factor I and the monoammonium salt of factor II)was administered in a single dose by gavage to each of two mice infectedwith Aspiculuris tetraptera and Syphacia obvelata (pinworms). The dosewas 500 milligrams of antibiotic A16886 per kilogram of individualanimal body weight, administered in a suspension of physiological salinecontaining 0.125 percent of methylcellulose as suspending agent. Acontrol group of six mice infected with Aspiculuris tetraptera andSyphacia obvelata was employed in the evaluation. Both groups weremaintained under normal laboratory conditions for forty-eight hoursfollowing the dosing of the treated group. All mice were then sacrificedand examined to determine the presence and numbers of pinworms, whichwere as reported in the following table:

                  TABLE XI                                                        ______________________________________                                                   Number of Pinworms per Animal (average                                        Aspiculuris tetraptera                                                                     Syphacia obvelata                                     ______________________________________                                        Control      35.0           41.0                                              Antibiotic A16886 at                                                          500 mg./kg.   4.5            0.5                                              ______________________________________                                    

EXAMPLE 11

In another evaluation, antibiotic A16886 (as a mixture of themonoammonium salt of factor I and the monoammonium salt of factor II)was mixed with standard mouse feed to obtain a plurality of treatedfeeds, containing antibiotic A16886 in concentrations of 0.005, 0.01 and0.05 percent by weight. The feeds were utilized as diets for separategroups of mice, five mice per group. About twelve hours after initiationof the feeding, the mice were infected with Ascaris lumbricoides var.suum ova. Another group of five mice was fed the non-medicated feed toserve as a control but was similarly infected at the same time withAscaris lumbricoides var. suum. All groups were fed their respectivefeed and maintained under normal laboratory conditions for a period ofeight days, at which time all mice were taken off feed. On the ninthday, all mice were sacrified and the lungs examined to determine thepresence, and if present, numbers, of lesions of Ascaris lumbricoidesvar. suum.

The level of antibiotic A16886 in the diet and the average number oflung lesions per animal in each group are set forth in the followingtable:

                  TABLE XII                                                       ______________________________________                                                           Average Number of Lung                                     Group              Lesions Per Group                                          ______________________________________                                        Control            2.0                                                        Antibiotic A16886 at 0.005 percent                                                               0.166                                                      Antibiotic A16886 at 0.01 percent                                                                0.33                                                       Antibiotic A16886 at 0.05 percent                                                                0.0                                                        ______________________________________                                    

EXAMPLE 12

Another evaluation was conducted under the procedures reported inExample II, except that the rates were varied. The results were asfollows:

                  TABLE XIII                                                      ______________________________________                                                           Average Number of Lung                                     Group              Lesions Per Group                                          ______________________________________                                        Control            2.2                                                        Antibiotic A16886 at 0.0005 percent                                                              1.2                                                        Antibiotic A16886 at 0.001 percent                                                               0.7                                                        Antibiotic A16886 at 0.1 percent                                                                 0.2                                                        ______________________________________                                    

EXAMPLE 13

In a further evaluation, the monoammonium salts of A16886I and A16886IIwere separately evaluated for the control of Ascaris lumbricoides var.suum. The evaluations were conducted in accordance with the procedurereported in Example 11 at a concentration in the diet of 0.05 percent.The results of the evaluations were as reported in the following table.

                  TABLE XIV                                                       ______________________________________                                                        Average Number of Lung                                        Group           Lesions Per Group                                             ______________________________________                                        A16886I         0.46                                                          A16886II        0.20                                                          Control         2.66                                                          ______________________________________                                    

Antibiotic A16886 can be produced by culturing a newly found andhitherto undescribed organism isolated from soil samples obtained fromSouth America.

The organism was isolated from the above soil samples by suspendingportions of the soil samples in sterile distilled water, and bystreaking the suspensions on nutrient agar. The seeded nutrient agarplates were incubated at about 25-35° C. for several days. At the end ofthe incubation period, colonies of the antibiotic A16886-producingorganism were transferred with a sterile platinum loop to agar slants.The agar slants were then incubated to provide suitable amounts ofinoculum for the production of antibiotic A16886.

The actinomycete used according to this invention for the production ofantibiotic A16886 has been difficult to classify in the genusStreptomyces because of its a typical sporophore morphology. However,date on cell wall analysis indicate that the culture should beconsidered a species in the Streptomyces genus. Accordingly, theorganism is treated as a new species and given the name Streptomycescalvuligerus.

This organism characteristically produces an extensive network of short,sympodially-branched aerial hyphae which eventually segment into spores.Short club-shaped side branches are formed which usually produce fromone to four spores each. No substrate conidia are produced. Electronmicrographs reveal smooth-walled spores. Cell wall preparations containthe L,L-isomer of diaminopimelic acid and glycine in addition to themajor constituents, aspartic acid, glutamic acid, and alanine. Sporesare gray en masse and primary mycelium is pale yellow to yellow-brown.No soluble pigment is produced. The culture has an optimum temperaturerange between 26° and 30° C. No growth occurs at 37° C. Morphologically,this culture resembles certain strains of Thermomonospora andMicromonospora.

The novel organism capable of producing antibiotic A16886 has beenplaced on permanent deposit without restriction as to availability withthe culture collection of the Northern Utilization Research andDevelopment Division, Agricultural Research Service, U.S. Department ofAgriculture (formerly Northern Regional Research Laboratories), Peoria,Illinois, 61604, and is available to the public under culture No. NRRL3585.

The characteristics of Streptomyces clavuligerus NRRL 3585 are given inthe following tables. The methods recommended for the InternationalStreptomyces Project (Shirling et al., "Methods for Characterization ofStreptomyces Species," Intern. Bull. Systematic Bacteriol. 16: 313-340)(1966) for the characterization of Streptomyces species have been usedalong with certain supplementary tests. Color names were assignedaccording to the ISCC-NBS method described by Kelly et al. in TheISCC-NBS Method of Designating Colors and a Dictionary of Color Names(U.S. Department of Commerce Circ. 553, Washington, D.C. 1955). Figuresin parenthesis refer to the Tresner and Backus color series (Tresner etal., "System of Color Wheels for Streptomyces Taxonomy," Appl.Microbiol, 11: 335-338 [1963]) and color tab designations areunderlined. The Maerz and Paul color blocks (Maerz et al., Dictionary ofColor (McGraw-Hill Book Co., Inc., New York, 1950) are enclosed inbrackets. Cultures were grown at 30° C. for 14 days unless notedotherwise.

                  TABLE XV                                                        ______________________________________                                        Property Observed                                                                             Characteristics of A16886                                     ______________________________________                                        Morphology      Sporophores are produced on an                                                extensive aerial mycelium and                                                 consists of networks of short,                                                sympodially-branched hyphae.                                                  Usually from one to four spores                                               are born on short club-shaped                                                 side branches. Eventually                                                     sporophores segment to form                                                   chains of spores. Spores are                                                  0.34-0.85μ × 0.85 × 3.3μ, averag-                           ing 0.64μ × 1.53μ. Electron                                       micrographs reveal smooth-walled                                              spores. Spores are not produced                                               in the substrate mycelium.                                    Cultural Characteristics on:                                                  ISP No. 2       Growth abundant, reverse grayish                              (Yeast-Malt ext. agar)                                                                        yellow [12K3]; aerial mycelium                                                abundant, dark gray (G) 3ih                                                   [21B1]; no soluble pigment.                                   ISP No. 3       Growth moderate, reverse pale                                 (Oatmeal agar)  yellow [11C1]; aerial mycelium                                                fair, white (W) b [27A1]; no                                                  soluble pigment.                                              ISP No. 4       Growth abundant, reverse grayish                              (Inorganic Salts-                                                                             yellow [12B2]; aerial mycelium                                Starch agar)    moderate, medium gray (GY) 2fe                                                [45A1]; no soluble pigment.                                   ISP No. 5       Growth fair, reverse pale yellow                              (Glycerol-asparagine agar)                                                                    green [10B1]; aerial mycelium                                                 fair, white (W) a; no soluble                                                 pigment.                                                      Tomato paste-oatmeal agar                                                                     Growth abundant, reverse gray-                                                ish yellow [11E4]; aerial                                                     mycelium moderate, light grayish                                              olive (GN) 11/2ig [21B1]; no                                                  soluble pigment.                                              Emerson's agar  Growth abundant, reverse pale                                                 yellow [11C1]; aerial mycelium                                                scant; no soluble pigment.                                    Bennett's agar  Growth abundant, reverse light                                                yellow [11J2]; aerial mycelium                                                abundant, dark grayish green                                                  (GN) 241/2ih [23A3]; no soluble                                               pigment.                                                      Czapek's agar   Scant growth                                                  Glucose-Asparagine agar                                                                       Growth moderate, reverse pale                                                 yellow green [10B1]; aerial                                                   mycelium fair, white (W) b                                                    [27A1]; no soluble pigment.                                   Tyrosine agar   Growth moderate, reverse pale                                                 yellow [10B2]; aerial mycelium                                                moderate, yellowish gray (GY)                                                 2dc [10A2]; no soluble pigment.                               Nutrient agar   Growth fair, reverse pale yellow                                              green [10B1]; aerial mycelium                                                 sparse, white; no soluble pigment                             Cultural Characteristics on:                                                                  Characteristics of A16886                                     ______________________________________                                        Calcium malate  Growth abundant, reverse pale                                                 yellow green [10B1]; aerial                                                   mycelium fair, white (W) a.                                   Action on milk  No coagulation; clearing in 17                                                days.                                                         Nitrate reduction                                                                             Negative                                                      Gelatin liquefaction                                                                          None                                                          Growth response to pH                                                                         pH 5.0-6.0 in optimum range for                               changes         growth; growth but no sporu-                                                  lation from pH 7.5-8.5.                                       Melanin production                                                            Peptone-iron agar and                                                         tryptone-yeast ext. broth                                                                     None                                                          Temperature requirements                                                                      Growth and sporulation good from                                              26-30°; no growth at 37° or above.              Major constituents of whole                                                                   L,L-diaminopimelic acid, glycine,                             cell hydrolysates                                                                             glutamic acid, aspartic acid,                                                 alanine, and leucine.                                         ______________________________________                                    

In Table XVI are set forth the results of carbon utilization testscarried out on organism NRRL 3585. In the table, the following symbolsare employed:

+ = growth and utilization

- = no growth, no utilization

(+) = probable utilization

(-) = questionable utilization

                  TABLE XVI                                                       ______________________________________                                        Carbon Utilization Pattern for NRRL 3585                                      Compound            Growth Response                                           ______________________________________                                        Carbon Utilization:                                                           L-arabinose         -                                                         rhamnose            -                                                         fructose            -                                                         D-xylose            -                                                         melezitose          (-)                                                       raffinose           -                                                         dextrose            -                                                         cellobiose          -                                                         maltose             +                                                         sucrose             -                                                         cellulose           -                                                         inositol            (+)                                                       mannitol            -                                                         Na glutamate        (+)                                                       ______________________________________                                    

As above noted, antibiotic A16886 can be produced by the cultivation ofNRRL 3585. The culture medium employed in producing antibiotic A16886 bycultivation of the above-identified organism can be any one of severalmedia, although as it is apparent from the above-described utilizationtests, the organism is capable of using only a few carbon sources underartificial culture circumstances. Those skilled in the art willunderstand that the organism is a complete medium may utilize carbonsources which in such artificial circumstances are not utilized.However, for economy production, maximum yield of antibiotic, and easeof isolation of the antibiotic, certain relatively simple nutrientsources are preferable. For example, the media which are useful in theproduction of the antibiotic include an assimilable source of carbonsuch as glucose, starch, glycerine, molasses, dextrin, and the like.

Preferred sources of carbon are glucose and glycerol. Additionally,employable media include a source of assimilable nitrogen such assoybean meal, corn steep solids, yeast, cotton-seed meal, beef extract,peptones (meat or soy), casein, amino acid mixtures, and the like.Preferred sources of nitrogen are peptones, soybean meal, amino acidmixtures, and the like. Among the nutrient inorganic salts which can beincorporated in the culture media are the customary salts capable ofyielding sodium, potassium, ammonium, calcium, phosphate, sulfate,chloride, carbonate, and like ions.

Minor elements necessary for optimum growth and development of theorganism used for the production of antibiotic A16886 can also beincluded in the culture medium. Such trace elements commonly occur asimpurities in the other constituents of the medium in amounts sufficientto meet the growth requirements of the actinomycetes employed in thisinvention.

The initial pH of the culture medium can be varied. However, it has beenfound desirable that the initial pH of the medium be between 6.5 and7.2. As has been observed with other actinomycetes, the pH of the mediumgradually increases throughout the growth period of the organism whilethe antibiotic is being produced, and may attain a level of from 6.7 to7.5 or above, the final pH being dependent at least in part on theinitial pH of the medium, the buffers present in the medium, and theperiod of time the organism is permitted to grow.

Submerged, aerobic cultural conditions are the conditions of choice forthe production of antibiotic A16886. For preparation of relatively smallamounts, shake flask and surface culture in bottles can be employed; butfor the preparation of large amounts, submerged aerobic culture insterile tanks is preferred. The medium in the sterile tank can beinoculated with a sporulated suspension; but because of the growth lagexperienced when a sporulated suspension is used as the inoculum, thevegetative form of the culture is preferred. By thus avoiding the growthlag, more efficient use of the fermentation equipment is realized.Accordingly, it is desirable first to produce a vegetative inoculum ofthe organism by incoculating a relatively small quantity of culturemedium with the spore form of the organism; and when a young, activevegetative inoculum has been obtained, to transfer the vegetativeinoculum aseptically to the large tank. The medium in which thevegetative inoculum is produced can be either the same as or differentfrom the medium utilized for the large-scale production of antibioticA16886.

The organism which produces antibiotic A16886 will grow over a widetemperature range between 25-37° C. Optimal production of A16886 seemsto occur at temperatures of 26-30° C. In general, maximum production ofthe antibiotic occurs within about 36-72 hours after inoculation of theculture medium.

As is customary in aerobic, submerged culture processes, sterile air isblown through the culture medium. For efficient growth of the organismand antibiotic A16886 production, the volume of air employed in the tankproduction of A16886 is from 0.2 to 0.4 volume of air per minute pervolume of culture. The preferred volume is 0.4 volume of air per minuteper volume of culture medium.

The concentration of antibiotic activity in the culture medium can befollowed readily during the fermentation period by testing samples ofthe culture medium for their inhibitory activity against the growth oforganisms known to be inhibited by the presence of antibiotic A16886.The organisms E. coli, Salmonella gallinarum, and Pseudomonassolanacearum have been found to be useful for this purpose. The testingof the samples can be carried out by the well-known turbidometric orcup-plate methods.

In general, maximum production of A16886 occurs within one to three daysafter inoculation of the culture medium in submerged aerobic culture orshake flask culture processes.

The antibiotic activity produced during the fermentation of A16886occurs primarily in the antibiotic broth. Accordingly, isolationtechniques employed in the production of A16886 are designed to permitmaximum recovery of the antibiotic from the broth. Thus, for example,mycelium and undissolved solids are removed from the fermentation brothby conventional means such as filtration or centrifugation, andantibiotic A16886 can be recovered from the filtered or centrifugedbroth by employing extraction or adsorption techniques.

For the recovery of A16886 by adsorption techniques, various adsorbentsand ion exchange resins can be used, for example, carbon, silica gel,alumina, microcrystalline cellulose, and ion exchange resins, includinginsoluble crosslinked polymer resins such as the insoluble crosslinkedpolystyrene known as Amberlite XAD-4 (marketed by Rohm & Haas Co.).Antibiotic A16886, as obtained from fermentation, may be in eitheramphoteric or salt form, depending upon fermentation conditions.Regardless of which form, it can be adsorbed onto one to the above orsimilar adsorbents from solution in a suitable solvent. The adsorbedantibiotic A16886 or salt can then be eluted from the adsorbent bysuitable elution techniques, such as by washing the adsorbent on whichthe antibiotic A16886 or salt thereof is adsorbed with a solvent. Wherethe elution is carried out by washing with a solution of, e.g., ammoniumformate or sodium acetate, the process results in elution of antibioticA16886 as the ammonium or sodium salt, respectively. Such salts arereadily converted back to antibiotic A16886 in conventional procedures.

Salts of antibiotic A16886 other than ammonium or alkali metal arepreferably prepared by conventional reaction of antibiotic A16886 inunmodified amphoteric form with the respective acid or base. Thus, inpreparing acid addition salts, antibiotic A16886 is reacted with aninorganic or organic acid. Representative suitable acids includehydrochloric acid, hydrobromic acid, hydriodic acid, sulfuric acid,phosphoric acid, acetic acid, benzoic acid, sulfamic acid, tartaricacid, citric acid, maleic acid, succinic acid, ascorbic acid, andglycolic acid.

Antibiotic A16886 also forms salts with cations by reaction of A16886 inunmodified amphoteric form with inorganic and organic bases and salts.Exemplary of these salts are ammonium and substituted ammonium salts;alkali metal salts, such as sodium, potassium, lithium, cesium, andrubidium salts; alkaline earth metal salts such as calcium, strontium,and barium salts; and copper, zinc, magnesium, and silver salts. In theinstance of organic bases, the identity of the base is not critical,although, in general, a base having a pH of, numerically, 3.0 or abovein water is preferred. Representative suitable organic bases includebenzylamine, methylamine, diethylamine, triethylamine, procaine,diisopropylamine, ethanolamine, cyclohexylamine, dicyclohexylamine,diphenylamine, di-n-butylamine, quinoline, and pyridylamine.

The salts of antibiotic A16886 which are pharmaceutically acceptable aregenerally preferred for pharmaceutical applications. However, all saltsare useful as intermediates in the production, separation, andpurification of antibiotic A16886. For therapeutic purposes,pharmaceutically acceptable salts are generally equivalent to antibioticA16886; however, particular salts are occasionally preferred due to afavorable property, such as solubility, conferred by the salt-formingmoiety.

In order to illustrate more fully the operation of the invention, thefollowing additional examples are provided by way of illustration.

EXAMPLE 14 SHAKE FLASK PRODUCTION OF ANTIBIOTIC A16886

A sporulated culture of Streptomyces clavuligerus NRRL 3585 was producedby growing the organism on a nutrient agar slant having the followingcomposition:

    ______________________________________                                        Dextrin                 10.00 g.                                              Yeast Extract            1.00 g.                                              Hydrolyzed Casein                                                             ("N-Z Amine-Type A,"                                                          Sheffield Chemical                                                            Company                  2.00 g.                                              Beef Extract             1.00 g.                                              Meer Agar (washed three                                                       times)                  20.00 g.                                              Deionized water          1 liter                                              ______________________________________                                    

The pH of the medium was adjusted to pH 7.0 by the addition of sodiumhydroxide.

The agar slant was inoculated with spores of Streptomyces clavuligerusNRRL 3585 and was incubated for 4-6 days at 30° C. The agar slant wasthen covered with sterile distilled water and gently scraped to removethe spores and cells as an aqueous suspension thereof. One milliliter ofthe resulting suspension was used to inoculate each 100 ml. portion of avegetative medium having the following composition:

    ______________________________________                                        Glucose                15.00 g.                                               Soybean meal           15.00 g.                                               Cornsteep solids        5.00 g.                                               Calcium carbonate       2.00 g.                                               Sodium chloride         5.00 g.                                               Deionized water         1 liter                                               ______________________________________                                    

The pH of the vegetative medium was adjusted to pH 6.7 by the additionof sodium hydroxide.

The vegetative inoculum was shaken for 24-48 hours at 30° C. on areciprocal shaker with a 2-inch stroke at 108 rpm. The inoculum soprepared was then utilized in the production of antibiotic A16886 asfollows.

A production medium was prepared having the following composition:

    ______________________________________                                        Soybean meal            15.00 g.                                              Casein                   1.00 g.                                              Sodium nitrate           3.00 g.                                              Glucose syrup (50 percent                                                     glucose)                20.00 g.                                              Tap water                1 liter                                              ______________________________________                                    

One hundred-milliter-portions of the production medium were placed in500-milliliter Erlenmeyer flasks which were sterilized at 120° C. for 30minutes. When cooled, each flask was inoculated with a five percentvegetative inoculum. The fermentation was shaken for 48-72 hours at25-30° C. on a rotary shaker operating at 250 rpm. During thefermentation, the medium was aerated with sterile air at a rate of 0.4v./v./min. Isolation was carried out essentially as reported hereinbelowin Example 22.

EXAMPLE 15

Antibiotic A16886 was produced according to the process of Example 14,but utilizing a production medium having the following composition:

    ______________________________________                                        Distillers Solubles (Nadrisol)                                                                        5.00 g.                                               Soybean flour (Nutrisoy 200D)                                                                         5.00 g.                                               Peanut meal             5.00 g.                                               Blackstrap molasses     5.00 g.                                               Oatmeal                 5.00 g.                                               Glycerol                10.00 g.                                              Tap water               1 liter                                               ______________________________________                                    

and utilizing instead of a rotary shaker a reciprocal shaker operatingat 108 strokes per minute.

EXAMPLE 16

Antibiotic A16886 was produced according to the process of Example 14,but utilizing a production medium having the following composition:

    ______________________________________                                               Oatmeal       20.00 g.                                                        Glycerol      10.00 g.                                                        Tap water      1 liter                                                 ______________________________________                                    

EXAMPLE 17

Antibiotic A16886 was produced according to the process of Example 14,but utilizing a production medium having the following composition:

    ______________________________________                                        Cottonseed flour      20.00 g.                                                Glycerol              10.00 g.                                                Glucose                5.00 g.                                                Tap water              1 liter                                                ______________________________________                                    

EXAMPLE 18

Antibiotic A16886 was produced according to the process of Example 14,but utilizing a production medium having the following composition:

    ______________________________________                                        Glucose                  20.00 g.                                             Soluble starch           10.00 g.                                             Peptone (Wilson's 159)   30.00 g.                                             Hydrolyzed casein ("N-Z amine-                                                type sold  sld by the Sheffield                                               Chemical Company)        4.00 g.                                              Magnesium sulfate heptahydrate                                                                         5.00 g.                                              Molasses, blackstrap     5.00 g.                                              Calcium carbonate        2.00 g.                                              Tap water                1100 ml.                                             ______________________________________                                    

EXAMPLE 19

Antibiotic A16886 was produced according to the process of Example 14,but utilizing a production medium having the following composition:

    ______________________________________                                        Glycerol               20.00 g.                                               Soy peptone           5.00 g.                                                 Calcium nitrate       2.00 g.                                                 Sodium chloride       0.50 g.                                                 Nadrisol              3.00 g.                                                 Tap water             1 liter                                                 ______________________________________                                    

EXAMPLE 20

Another sporulated culture of Streptomyces clavuligerus NRRL 3585 wasproduced by growing the organism on a nutrient agar slant. The slant inthis instance had the following composition:

    ______________________________________                                        Dextrin                   10.00 g.                                            Yeast extract            1.00 g.                                              Hydrolyzed casein ("N-Z Amine-                                                Type A") (Sheffield Chemical                                                  Company)                 2.00 g.                                              Beef extract             1.00 g.                                              Calcium chloride heptadhydrate                                                                         0.01 g.                                              Meer agar                 20.00 g.                                            Deionized water          1 liter                                              ______________________________________                                    

The pH of the medium was adjusted, by addition of sodium hydroxide, to7.0.

The agar slant was inoculated with spores of Streptomyces clavuligerusNRRL 3585 and incubated for 7-10 days at 30° C. The agar slants werethen scraped to remove spores to which were added 2.0 ml. of sterilebeef serum. Of the resulting serum spore suspension, 0.1 milliliter wasthen transferred to a sterile lyophile tube and freeze-dried in the formof pellets.

The pellets thus obtained were used to inoculate a vegetative mediumhaving the following composition:

    ______________________________________                                        Glycerol               10.00 g.                                               Sucrose                20.00 g.                                               Nutrisoy grits         15.00 g.                                               Amber BYF 300           5.00 g.                                               Tryptone                5.00 g.                                               Potassium biphosphate   0.20 g.                                               Tap water              1 liter                                                ______________________________________                                    

The pH of the medium was 6.2 and was adjusted to 6.5 with sodiumhydroxide.

EXAMPLE 21 PILOT PLANT PRODUCTION OF ANTIBIOTIC A16886

To a 40-liter stainless steel fermentor were added 24 liters of a mediumhaving the following composition:

    ______________________________________                                        Antifoam A (an anti-foaming agent                                             sold by the Dow Corning Company)                                                                      5.00 g.                                               Starch                  1125.00 g.                                            Nadrisol               125.00 g.                                              Soybean flour grits    500.00 g.                                              Glycerol               187.50 g.                                              N-Z amine A            125.00 g.                                              Ferrous sulfate heptahydrate                                                                          2.50 g.                                               Cold tap water         to 24 liters                                           ______________________________________                                    

The initial pH was 5.9 and was adjusted to pH 6.5 with approximately 20ml. of 5N sodium hydroxide. The medium was sterilized for 30 minutes at120° C. and 15-18 psig., cooled, and then inoculated with a five percentvegetative inoculum produced as in Example 20. The fermentation wascarried out at 30° C. for 66 hours, aerated with sterile air at the rateof 0.35 v./v./min., and agitated by a mechanical stirrer operated at 420revolutions per minute. The terminal pH was 6.3.

Antibiotic A16886 was recovered from the broth following the isolationprocedure set forth in Example 22.

EXAMPLE 22 ISOLATION OF CRUDE ANTIBIOTIC A16886 as MONOAMMONIUM SALT

Approximately 75 liters of broth obtained as reported in Example 21 werefiltered with the aid of Hyflo Super-Cel (a diatomaceous earth sold byJohns-Manville Products), 5 grams per 100 milliliters. The brothfiltrate was passed over a 9.5 cm. × 130 cm. column packed with 8 litersof carbon (Pittsburgh 12 × 40), at the rate of 60 ml. per minute. Thecolumn was washed with 10 liters of deionized water (pH 5.2) and theactivity adsorbed on the carbon was removed by passing 50 percentaqueous acetone over the column. The fractions containing the activitywere combined, concentrated in vacuo to remove acetone, and applied to a9.5 cm. × 140 cm. column packed with Dowex 1-X1 (a strong basic anionexchange resin sold by the Dow Chemical Co.), in the formate cycle. Thecolumn was washed with 10 liters of deionized water, and the activitywas removed with 0.1M ammonium formate. The active fractions werecombined, and passed over a 9.5 cm. × 100 cm. column packed with carbon(Pittsburgh 12 × 40), at the rate of 60 ml. per minute. The column waswashed with water; and the activity was then eluted with 1/4acetone:water at 60 ml. per minute, yielding 15 two-liter fractions, andthereafter with 1:1 acetone:water, yielding 18 one-liter fractions. Theactive fractions were combined, concentrated in vacuo to remove acetone,and lyophilized.

Forty grams of combined lyophilized preparations, each preparation asdiscussed above, were extracted with 4 liters of methanol by stirringmagnetically for 16 hours; the methanol insolubles were filtered off andthe methanol soluble portion precipitated with 5 volumes of acetone. Theprecipitate was separated by filtration and dried. Yield was 20.6 grams.

This preparation was dissolved in a minimum of water and applied to a5.8 cm. × 120 cm. column packed with dextran (Sephadex G-25) at a rateof 1 ml. per minute. The activity was eluted with deionized water, andthe active fractions were combined and lyophilized.

Ten grams of material obtained as described was dissolved in 256 ml. ofacetonitrile:water (55:45) and applied to a 5.5 cm. × 85 cm. columnpacked with silica gel prepared in acetonitrile:water (7:3) solvent.Application was at the rate of 3 ml. per minute. After application ofthe sample, the column was eluted with acetonitrile:water (7:3) at 5 ml.per minute flow rate. The most active fractions were combined,concentrated to dryness in vacuo, and lyophilized.

Material thus obtained was a mixture of the monoammonium salts offactors I and II; it was used in the antibacterial and anthelminticevaluations reported above, as well as in separation and purificationprocedures reported below.

EXAMPLE 23 SEPARATION OF FACTORS I AND II OF ANTIBIOTIC A16886MONOAMMONIUM SALT

Ten grams of a preparation prepared as described in Example 22 weredissolved in 197 milliliters of acetonitrile:water (55:45) and appliedto a 4.0 cm. × 130 cm. column packed with 1.6 liters of microcrystallinecellulose (Avicel) in acetonitrile:water (7:3). The column was loaded at1 ml. per minute, and eluted at 2 ml. per minute with acetonitrile:water(7:3). The elution was followed by assay and paper chromatography. As aresult of the elution, a plurality of fractions was obtained, eachcontaining predominantly one factor. The fractions of the respectivefactors were pooled and lyophilized.

EXAMPLE 24 PREPARATION OF ANTIBIOTIC A16886I HYDROCHLORIDE

Antibiotic A16886I monoammonium salt, prepared and separated inaccordance with the foregoing examples (200 milligrams), was dissolvedin 2 ml. of water. The pH of the solution initially was 5.30; it wasadjusted to pH 2.0 with 1N HCl and then concentrated under reducedpressure, to near dryness. Water (0.5 ml.) and methanol (1.5 ml.) wereadded, and the desired hydrochloride salt precipitated by the additionof ten volumes of acetone. The precipitated antibiotic A16886Ihydrochloride was removed by filtration, washed with acetone, and dried.Analysis showed 4.50 percent chlorine. Electrometric titration of theproduct in a 66 percent dimethylformamide-water solution at an initialpH of 5.0 revealed the presence of three titratable groups: pK'a₁ = 3.6;pK'a₂ = 5.2; and pK'a₃ = 10.8.

EXAMPLE 25 PREPARATION OF ANTIBIOTIC A16886I DISODIUM SALT

Antibiotic A16886I monoammonium salt (200 mgm.), prepared and separatedin accordance with the preceding examples, was dissolved in 2 ml. ofwater. The initial pH was 5.30. The solution was then adjusted to pH10.50 with 2.5N NaOH and concentrated, under reduced pressure to neardryness. Methanol (1.5 ml.) was added and the salt precipitated with tenvolumes acetone. The precipitated antibiotic A16886I disodium salt wasremoved by filtration and washed with acetone, and dried. Analysisshowed 6.79 percent sodium.

EXAMPLE 26 PREPARATION OF ANTIBIOTIC A16886 IN ACID FORM

Two hundred milligrams of a mixture of the monoammonium salt ofantibiotic A16886I and the monoammonium salt of antibiotic A16886II weredissolved in 40 milliliters water, and 6 milliliters of Dowex 50W-X12(H+) resin (sold by the Dow Chemical Co.) were added. The mixture wasstirred for thirty minutes and filtered, using a medium sinter glassfunnel, and the filtrate (pH 2.65) was lyophilized.

Electrometric titration in a 66 percent dimethylformamide-water solutionat an initial pH of 4.30 revealed the presence of three titratablegroups: pK'a₁ = 4.2; pK'a₂ = 5.6; and pK'a₃ = 10.4.

We claim:
 1. A method for the inhibition of a plant-pathogenic bacteriumwhich comprises applying to a plant exposed to the bacterium an amountof7-(5-amino-5-carboxyvaleramido)-3-(carbamoyloxymethyl)-3-cephem-4-carboxylicacid which is effective to inhibit the growth of the bacterium.
 2. Thecomposition suitable for inhibiting plant-pathogenic bacteria comprising(1)7-(5-amino-5-carboxyvaleramido)-3-(carbamoyloxymethyl)-3-cephem-4-carboxylicacid, in an amount which is effective to inhibit the growth of aplant-pathogenic bacterium, and (2) a surface-active agent.